Transcription factors are central players in the regulatory networks that control differentiation in all types of precursor cells and stem cells. Itis well known that transcription factors regulate gene expression, but it is unclear how they regulate differentiation. For instance, in some cases a given transcription factor promotes cell differentiation in one context, and prevents it in another. Melanocytes are an ideal model cell type to explore cellular developmental events because their differentiation is easily scored, they are not essential for life, and loss of melanocyte stem cells in mammals is readily apparent by the consequent hair graying. Moreover, mutations in genes encoding transcription factors and other regulatory molecules cause developmental disorders of pigmentation (e.g., Hirschsprung's disease, Tietz syndrome, Waardenburg syndrome types I- IV, piebaldism). Our long-term objective is to understand the regulatory network governing developmental events in the melanocyte lineage. Here we focus on the transcription factor Activator Protein 2 family (TFAP2A-E). TFAP2A appears to prevent differentiation of melanocyte stem cells, because dominant mutations in TFAP2A cause Branchio Oculo Facial Syndrome (BOFS), which features premature hair graying. By contrast, TFAP2A and its paralog TFAP2E appear to promote differentiation of embryonic melanocytes, as shown by our work in zebrafish. We hypothesize that within melanocyte precursors, TFAP2 proteins function redundantly, and in parallel with microphthalmia-associated transcription factor (MITF), a central regulator of melanocyte biology, to activate genes that effect melanocyte differentiation. To test this model we will conduct knock-down/add-back studies in zebrafish, generate appropriate tissue- specific double mutants in mice, and conduct functional analysis of the promoter of a gene mutually activated by TFAP2 proteins and MITF in cultured in human melanocytes. Next we will examine how TFAP2A functions to maintain melanocyte stem cells (MSC). We hypothesize that in this context, the key function of TFAP2A is to activate KIT. To test this notion we will use zebrafish again for epistasis tests, examine hair graying in a mouse with neural-crest-specific deletion of TFAP2A, and assess functional properties of TFAP2A variants associated with hair graying in BOFS. Finally, we will conduct systematic analyses of potential regulators of TFAP2A expression in melanocytes, and a global analysis of TFAP2A transcriptional targets in melanocytes, which together will position TFAP2A into a regulatory network in melanocytes. Our studies will shed light on novel genetic and molecular networks that underlie normal and pathological development of melanocytes. The new knowledge generated from this work will be of value specifically in the etiology of Waardenburg syndrome and BOFS. More generally, it will illuminate the transcriptional regulation of differentiation in a tractable model cell type.